1. Please state project objectives and what work was done this quarter to address them:
Objective 1: Evaluate currently available registered insecticides in Florida citrus against DRW.
Sub-objective 1: Evaluation of traditional insecticides.
Insects. Diaprepes root weevil (DRW) larvae were obtained from a culture at University of Florida’s Citrus Research and Education Center (CREC) in Lake Alfred, FL. This culture was periodically supplemented with collections of adult beetles from citrus groves in Florida. Larvae were reared on an artificial diet developed by Beavers (1982) using procedures described by Lapointe and Shapiro (1999). Larvae used in experiments were 3rd instars.
Morality of larval Diaprepes was determined using a soil assay procedure previously established for evaluating insecticide against the ground inhabiting stage of this pest (Hamlen et al. 1979). Candler sand was used as the substrate and was sieved using a 6-inch N.B.S. #20 sieve (pore size, 841 M) to remove larger particulate matter. The soil was autoclaved and allowed to air dry. Afterward, 9 ml of DI water was added back to the soil to reach approximately 12% moisture content. Approximately 25g of soil was then added to bioassay columns to a depth of 3cm. The columns were constructed from 50mL polystyrene tubes (12.0 cm height, 3.0 cm diameter) that were similar to those described previously by Hamlen et al. (1979). The objective of this experiment was to evaluate insecticides and associated application rates used against Asian citrus psyllid (ACP) to determine if they were also effective against the larval stage of Diaprepes.
The insecticides tested and associated rates are given in Table 1. Each insecticide formulation was added to DI water to yield concentrations of 0, 0.27, 2.7, 27ppm and field rate (270 ppm). A total of 1.040 ml of each concentration (or treatment) was pipetted uniformly onto the soil in the bioassay columns. Deionized water alone was used as the negative control. Five neonate Diaprepes larvae were scattered on the surface of the soil per replicate bioassay chamber and a total 12 replicate chambers were established per treatment evaluated. Bioassay units were kept in an incubator held at 25 ± 2 ºC, 50±10 % RH, and 14:10 L:D photoperiod. After 48hr, the number of living and dead Diaprepes larvae recovered in the containment cell at the base of the bioassay column was recorded. Also, soil was thoroughly excavated under a stereo microscope to find any remaining larvae in each chamber to determine morality. The relationship between chemical concentration and larval recovery was determined by probit analysis. Mortality data using field rates were analyzed using a generalized linear model (GLM) with binomial distribution followed by Tukey estimated marginal means, using the package emmeans in R for post hoc comparisons at = 0.05.
We previously reported that all of the insecticides evaluated in Table 1 cause > 90% mortality of Diaprepes larvae in soil. With Exirel, Sivanto, Danitol, and Delegate applied at field rates, we observed 100% mortality of Diaprepes larvae.
Results and next steps
During the past quarter, we have made further progress with establishing LC50 values for the other insecticides (Table 2). These baseline values will allow for monitoring of possible changes in insecticide susceptibility among DRW populations exposed to these insecticides over time. Also, this provides is with an assessment of the relative level of toxicity of various insecticides against DRW. We will continue establishing LC values for all of the insecticides that we have found to be effective against DRW.
Sub-objective 2: Evaluation of B. thuringiensis tenebrionis (Btt) activity against DRW.
Bioassays with DRW larvae. Bioassays were conducted to evaluate survival of DRW neonates and 5-week-old larvae after exposure to bacterial suspensions of B. thuringiensis tenebrionis (Btt). Five concentrations of Btt (in sterile distilled water) and a water control were prepared as described previously. There were fifteen replicates per treatment for neonates and five replicate per treatment for 5-week-old larvae. A total of 375 and 25 DRW neonate and 5-week-old larvae were starved for 3 -4 h prior to evaluation. Each treatment received 0.8 g of potato that had been weighted using an electronic balance (Mettler AE 160). Prior to larval exposure, the potato was submerged for 20 min in either the bacterial suspensions being tested or sterile distilled water, and then air-dried at ambient temperature for 10 min. Larvae were placed in Petri dishes containing potato pieces. Larvae that did not show signs of life after prodding with a needle were considered dead. Using this criterion, the number of dead and live insects was recorded after six weeks of exposure to treated potato.
Evaluation of Btt using artificial diet. DRW diet was heated to 90°C for 15 min., covered with foil, and allowed to cool to 56°C in a heated water bath before incorporating treatments. A commercial preparation of B. thuringiensis subsp. tenebrionis (CX-2330 85% [AI]) was incorporated into the diet at rates of 0, 0.3, 3, 300, and 3000 ppm (µg AI/ml diet). Treatments were incorporated into the agar-laden diet with the aid of a heated, stirrer plate. The resulting mixtures were pipetted into diet cups (15 ml diet per cup), allowed to solidify and cool to room temperature, and then were closed with a lid. All steps after heating of the diet were performed in a laminar flow, clean bench to avoid contamination.
Neonate larvae: The biological activity of CX-2330 was evaluated initially against neonate larvae (1st instar) exposed to treated artificial diet. The treatments included the 5 rates of treatment-incorporated diet described above and there were 10 replications of each treatment. A treatment comprised 10 diet cups, each infested with 5 neonate larvae. The numbers of dead larvae in each diet cup were assessed after 6 weeks of exposure to treated diet.
Five-week-old weevil larvae: The activity of CX-2330 also was evaluated against 5-week-old weevil larvae. Treatments were prepared as above and replicated 10 times. Each treatment comprised 10 diet cups, each containing 1 larva. Mortality and weights of emerging adults were recorded after 6 weeks of exposure to treated diet (Weathersbee et al., 2002).
Evaluation of Btt applied to citrus seedlings in greenhouse experiment. An additional experiment was conducted to determine the effect of CX-2330 suspensions applied as soil treatments against neonate and 5-week-old larvae feeding on potted citrus roots. The citrus plants used in the study were six-month-old old Cleopatra mandarin (Citrus reshni Hort., ex Tan.) rootstock potted in 588.75 cm3 containers of soil-based mix (three parts Peat Moss, two parts Coco Peat, one part Perlite, and one part gravel-sand-soil mixture). The treatments included 5 rates of CX-2330 (0, 3.0, 30, 300, and 3000 ppm [(g AI/ml DI water]) applied as 50 ml suspensions to the soil of each container. There were 4 replications of each treatment. A treatment comprised 1 potted citrus plant infested with 20 and 5 neonates and 5-week-old immediately after the drench application. All treatments were maintained in a growth chamber at 26°C with a photoperiod of 14:10 (L:D) h. The numbers and weights of surviving larvae were assessed after 6 weeks (Weathersbee et al., 2002).
Two-choice test evaluating effect of Btt on DRW egg laying and feeding inhibition. Six-month-old sour orange seedlings purchased from a nursery (Zimmerman’s Tropicals Nursery, FL, USA) were used to evaluate the effects of B. thuringiensis subsp. tenebrionis (Btt) on DRW feeding. Seedlings were planted in 9-cm dia. plastic pots filled with 588.75 cm3 sand acclimated in a growth chamber at 26°C with a photoperiod of 14:10 (L:D) h, for several weeks prior to the experiment.
Plants were treated with CX-2330 or water controls and presented to two mating pairs of DRW in choice bioassay (10 replications of each treatment in each type of assay). Choice tests compared a untreated citrus seedlings with citrus seedlings inoculated with CX-2330 suspension. Bioassays were run in square arenas (10 × 10 × 10 cm) at approximately 27 °C and 40% RH. Numbers of eggs laid and feeding on leaves were recorded after 10 and 4 days, respectively (Addesso et al., 2023).
Data Analyses and Statistics. Data were analyzed by the General Linear Models Procedure, and differences among treatment means were determined by LSD’s studentized range test (SAS Institute 1990). Differences among means were considered significant at a probability level of 5 percent (P = 0.05). Data from the two-choice feeding tests were subjected to a t-test (IBM SPSS Statistics 26).
Results
The survival of DRW larvae exposed as neonates to diet treated with CX-2330 was significantly reduced (Df= 70, 4; F value= 3.41; P= 0.0132; Df= 45, 4; F value= 11.43; P<.0001) after 6 weeks as compared with controls. However, diet treated with CX-2330 did not significantly increase (Df= 20, 4; F value= 2.09; P= 0.1200; Df= 20, 4; F value= 12.86; P<.0001) mortality of 5-wk-old larvae as compared with the control.
Mortality of neonates on potted citrus was increased (Df= 15, 4; F value= 1.48; P= 0.2569) by soil treatment with CX-2330 as compared with controls. Larval survival was (P = 0.05) lower in all treatments as compared to the controls after 6 weeks of exposure to treated soil and citrus roots (Table 3). Also, the fresh weights of surviving larvae were reduced (Df= 15, 4; F value= 1.83; P= 0.1748) by treatments indicating that larval feeding or nutrient assimilation may have been reduced.
Plants treated with the B. thuringiensis subsp. tenebrionis formulation lost less leaf tissue compared to control plants after 4 days (t Value= -0.417, p <.691) (Fig. 1). There was no significant difference in the number of eggs laid on plants treated with CX-2330 as compared with the control (t Value= -0.179, p <.864) (Fig. 2).
Interpretation and next steps
During this initial replicate of the experiment investigating movement of DRW between adjacent natural areas and citrus groves, we found no evidence that the natural area serves as a significant source of beetle infestation. In fact, the data suggest the opposite hypothesis that the grove contains an established resident populations of DRW that will require eradication with combined management of adults in the trees and larvae in the soil. However, these data represent only a single replicate both spatially and temporally and will need to be repeated before conclusions can be drawn.
If Diaprepes management in this grove does not depend on managing migrating insects (edge effects), but rather a resident population, the identification of hot spots in the grove might reveal soil properties that promote them. In Florida, weevil abundance varies by ecoregion and there is evidence that regionally adapted entomopathogenic nematodes (EPN) contribute to the weevil spatial pattern on the peninsula. Weevils also typically persist at higher, more damaging levels at specific locations within some orchards for unknown reasons. We hypothesize that soil properties driving soil food webs contribute to these local patterns. The ongoing survey has monitored tree condition and DRW adults in the tree canopy and as they emerge from the soil at 94 sites arranged in a grid pattern. Soil physico-chemical properties were characterized from soil samples taken from the sites in May 2023. DNA was also extracted from soil organisms recovered from 250 cm3 subsamples using sucrose centrifugation. The extracted DNA was used to measure (qPCR) populations of two entomopathogenic nematode species detected previously in this grove. It was also used to map the spatial distribution in the soil of species of fungi, mites, springtails, insects, nematodes, and bacteria using ITS2 rDNA, C0I mtDNA and 16S rDNA metabarcoding. To date only the ITS2 data (primarily fungi) has been recovered.
Objective 2: Within field
As reported previously, the weevils trapped in the tree canopies during 12 months were significantly aggregated, as measured by Spatial Analysis by Distance Indices (SADIE Ia index), and occurred primarily at the grove edge that confirmed the grower's previous observations. The weevil pattern was strongly associated (SADIE Xindex) with the pattern of tree death during spring 2023 and dissociated with that of elevation. The EPNs Steinernema diaprepesi were ubiquitous in the orchard, whereas Heterorhabditis indica were detected in just 14% of samples. The patterns of both EPN were significantly associated with that of dead trees, and the S. diaprepesi pattern was associated with that of weevils in the canopy, but dissociated with that of weevils emerging from soil. Assuming that high numbers of weevils in soil are the primary cause of tree mortality at this site, these relationships are consistent with density dependent regulation of weevils and EPN.
The edaphic properties and the microarthropod, nematode, fungal and bacterial components of soil food webs identified from metabarcoding will be employed in ordinal analyses to identify potential drivers of weevil and EPN abundance in local microhabitats. Similar inductive approaches involving fewer system components have identified soil features such as texture, moisture and pH that are amenable to manipulation in ways subsequently shown to modulate EPN persistence and efficacy. To date, the insect pathogens Beauveria bassiana and Hirsutella sp. were significantly dissociated from weevils because they clearly inhabit higher elevation sites than D. abbreviatus. In combination with 4 edaphic properties, the Hirsutella entomopathogen explained significant variability in D. abbreviatus counts when subjected to redundancy analysis (see Figure 3). By contrast, only elevation explained variability in the identified fungal entomopathogens. Recovery of the genetic barcode sequences among the 16S and COI genes will permit a more comprehensive analysis of the role of the soil food web in driving the weevil spatial patterns and will be described and discussed in the next report.
2. Please state what work is anticipated for next quarter: We will continue evaluation of tradition and Bt insecticidal formulations as well as continue the trapping study to describe the population demographics throughout the year. We anticipate that field populations of adults should be reduced over the coming months and therefore are uncertain if we will be able to run a second mark-recapture assay this coming quarter or not.
3. Please state budget status (underspend or overspend, and why): We are slightly underspent due to challenges in onboarding staff at the start of the project. We will request to carry over the funds to enable research to continue unimpeded and to ensure we have sufficient funds to support staff to replicate the mark-recapture assays on the east coast.
4. Please show all potential commercialization products resulting from this research, and the status of each: na